Composition to be administered to a living being and method for marking agents

ABSTRACT

The invention relates to compositions to be administered to a living being, a method for marking agents that are administered to living beings, the use thereof, and an accelerated test.

The invention relates to a composition to be administered to a livingbeing and to methods of labeling agents which are administered to livingbeings. The invention furthermore relates to uses of said compositionand said method of the invention and to an accelerated test.

The labeling of substances is nowadays becoming more and more important.Whether it is fossil fuels to be labeled in order to be better able tomonitor pollution possibly caused by said fossil fuel by detecting itsorigin, or whether it is the labeling of medicaments, for examplevaccines, comprehensive detection of their origin with respect to bothtime and geography and also of their sale, their transport etc. isdesired in all cases. A solid chain of detection is required inparticular, for example, in the case of vaccines administered to humansand/or animals. In the past, this was attempted by labeling thecorresponding packaging of the vaccine in a complicated manner. However,this “external labeling” has obvious disadvantages, since an unambiguousclassification cannot be guaranteed, after the medicament, vaccine etc.have been administered or in the case of non-authorized replacement ofthe packaging, falsification of the label.

A, compared to this, more advantageous type of labeling is directlabeling of the medicament/vaccine etc. to be administered itself andnot of its packing. This kind of “internal” labeling is proposed, forexample, in DE 198 47 118, where in addition an immunogen which isharmless to the particular organism is admixed to the agent to beadministered, which immunogen then elicits in said organism an immuneresponse, in particular the formation of antibodies or T-cells. Proposedimmunogens are: keyhole limpet hemocyanin (KLH) from Megathuracrenulata, green fluorescent protein (GFP) from Aequoria victoria,inactive snake toxins and viral proteins. Advice on the use ofvirus-like particles cannot be found in DE 198 47 118. The immunogensdisclosed in the prior art have the disadvantage that either they do notelicit any long-lasting immune responses after a single administration(e.g. KLH) and/or their preparation is clearly too expensive in order tobe able to use them commercially on a larger scale (e.g. KLH or GFP).Owing to the time-limited traceability of the antibody responsefollowing a single KLH injection, for example, the non-responder rate inpigs increases dramatically during the second half of a fattening period(average fattening period in Germany: 20-24 weeks). Thus, there is afundamental uncertainty in that it is not possible to detectsubsequently, whether the animal/the patient to which whom the agentassociated with KLH was administered simply did not exhibit any immuneresponse (i.e. is a “non-responder”) or whether the agent/the vaccinewas administered incorrectly (referred to as “non-compliance”).

Siray et al., 1998, Virus Genes 18, 39-47 disclose the possibility ofexpressing the VP1 protein of the polyoma virus from Syrian hamster(=golden hamster) in the form of an insoluble fusion protein in E. coliand the suitability of preparations of this kind for generating VP1antisera in rabbits. However, the antiserum generated by Siray et al.showed crossreactivity to VP1 of other species, thus renderingimpossible its use for labeling administered agents or agents to beadministered.

Gedvilaite et al., 2000, Virology, 273, 21-35 describe the formation ofSyrian hamster chimeric VLPs which are insoluble in aqueous solution andin which foreign epitopes have been incorporated. Gedvilaite et al.describe the use of these VLPs as vehicles for foreign vaccinesincorporated in the form of epitopes into said VLPs. The study stressesthe necessity of using complete virus-like particles (VLPs) in order toincrease the epitope density and thereby to generate an immune responsein the first place. The advantages of VLPs expressed in yeast are alsonoted, since these are the only endotoxin-free VLPs. In addition, thepossibility of using Syrian hamster VLPs as possible carriers of geneconstructs in gene therapy is mentioned.

It is the object of the present invention to provide a compositionwhich, with respect to its labeling, can be prepared in a simple andinexpensive manner, which is absolutely harmless to the animal/thepatient, which furthermore, with regard to its single label, produces insaid animal/said patient a long-lasting immune titer higher orlonger-lasting than the titer observed in connection with previouslabels, and which, owing to its non-existing non-responder rate, rendersa distinction between non-responder reaction and non-complianceunnecessary.

This object is achieved by a composition to be administered to a livingbeing, comprising:

a) an agent selected from the group comprising medicaments, vaccines andstored blood, and

b) at least one type of protein complex, said protein complex being asingle viral capsomer soluble in aqueous solution.

Preference is given to the protein complex being a single viral capsomerwhich is soluble in aqueous solution and which is an aggregated sandwichwith other single viral capsomers soluble in aqueous solution.

In one embodiment, the protein complex is a monomeric viral capsomer.

In this connection, the term “monomeric” refers to the absence of anassociation with other viral capsomers.

In another embodiment, the protein complex is a viral capsomer whichtogether with other viral capsomers forms an unspecific association ofat least two capsomers. Said association varies in size as a function ofthe external conditions (e.g. buffer, temperature, concentration) andmay comprise more than 20 capsomers. An association of this kind formsspontaneously under certain conditions and does not need any separatereconstitution step. An association of this kind is also referred tohere as “aggregate” which, however, does not form a complete VLP orviral capsoid.

In one embodiment, the protein complex is not in the form of a viralcapsoid. In one embodiment, the protein complex is soluble in aqueoussolution.

Preference is given to the viral capsomer being produced recombinantly,particularly preferably in a prokaryotic expression system, inparticular in E. coli.

In one embodiment, the viral capsomer is derived or can be obtained froma virus selected from the group of non-enveloped viruses, comprisingPapovaviridae, in particular polyoma and papilloma viruses,Iridoviridae, Adenoviridae, Parvoviridae, Picornaviridae, in particularpolio viruses, Caliciviridae, Reoviridae and Birnaviridae.

Preferably, the viral capsomer is derived or can be obtained frompolyoma virus, in particular murine polyoma virus.

In one embodiment, the viral capsomer is a pentamer, hexamer orheptamer.

The terms “pentamer”, “hexamer”, “heptamer” refer to a single viralcapsomer being composed of a plurality of viral capsomer proteins.Accordingly, a pentamer is a viral capsomer composed of five viralcapsomer proteins, a hexamer is a viral capsomer composed of six viralcapsomer proteins, etc.

In a preferred embodiment, the viral capsomer is a pentamer of murinepolyoma virus VP1 or is a pentamer of murine polyoma virus VP1 inassociation with murine polyoma virus VP2, or is a pentamer of murinepolyoma virus VP1 in association with murine polyoma virus VP3, or is acombination of the aforementioned possibilities. The term “pentamer . .. in association with VP2/3 . . . ” refers to the combination of apentamer with a molecule VP2/3 . . . .

In an association of a VP1 pentamer with VP2, preference is given to VP2being associated with at least one peptide. Preferably, the peptide isas defined below.

Particular preference is given to the viral capsomer being a pentamer ofmurine polyoma virus VP1.

In one embodiment, the viral capsomer is derived or can be obtained froma virus selected from the group of enveloped viruses, comprisingPoxviridae, Herpesviridae, Hepadnaviridae, Retroviridae,Paramyxoviridae, Sendaiviridae, Orthomyxoviridae, Bunyaviridae,Arenaviridae, Toroviridae, Togaviridae, Flaviviridae, Rhabdoviridae andFiloviridae.

In one embodiment, the viral capsomer does not derive or cannot beobtained from a virus which enters the organism of the living being inthe form of a vaccine or medicament or via the food chain or, undernormal conditions of life of said living being, via the environmentand/or to which antibodies are produced in said living being undernormal conditions of life, it being preferred that the virus-likeparticle does not derive or cannot be obtained from a virus selectedfrom the group comprising CSF virus (swine fever virus), foot-and-mouthdisease virus, PPV (porcine parvovirus), influenza virus, in particularinfluenza A virus, bovine leukemia virus (EBL virus), bovine herpesvirus (BHV1), bovine viral diarrhea virus (MD virus), bovine polyomavirus (BpyV), rotavirus, porcine herpes virus 1, pseudorabies virus,PRRS virus and TGE virus.

In one embodiment, the viral capsomer is associated with at least onepeptide (association of viral capsomer and peptide).

Preference is given to the association of viral capsomer and peptidebeing soluble in aqueous solution.

In one embodiment, the peptide is immunogenic when administered to aliving being, preference being given to said peptide being a peptideeliciting a B-cell response.

In one embodiment, the at least one peptide has been insertedrecombinantly into the viral capsomers.

Preferably, the at least one peptide has a sequence derived from avirus, a prokaryotic cell or a eukaryotic cell. In one embodiment, theat least one peptide has a sequence which is of artificial origin.

In one embodiment, the peptide comprises no more than 5-35 amino acids,preferably no more than 5-20 amino acids and more preferably no morethan 5-15 amino acids.

In one embodiment, the peptide is selected on the basis of one or moreof the following criteria: probability of being located on the surfaceof a protein structure (surface probability), flexibility, hydropathyand antigenicity, with the peptide preferably having high surfaceprobability, flexibility and antigenicity in conjunction with lowhydropathy. When selecting the peptide in this way, one or more of thefollowing methods may be applied, for example: [Boger, J., Emini, E. A.& Schmidt, A. Reports on the Sixth International Congress in Immunology(Toronto) 1986 p. 250; Chou P Y, Fasman G D. Adv Enzymol Relat Areas MolBiol. 1978; 47:45-148; Emini E A, Hughes J V, Perlow D S, Boger J. J.Virol. 1985 September; 55(3): 836-9; Garnier, J. Osguthorpe, D. J. andRobson, B. J. Mol. Biol. 1978, 120, 97-120; Hirakawa H, Muta S, KuharaS. Bioinformatic 1999 February; 15(2):141-8; Hopp T P, Woods K R. ProcNatl Acad Sci USA. 1981 June; 78(6): 3824-8; Jameson B A, Wolf H. ComputAppl Biosci. 1988 March; 4(1): 181-6; Janin J, Wodak S. J Mol Biol. 1978Nov. 5; 125(3): 357-86; Kyte J, Doolittle R F. J Mol Biol. 1982 May 5;157(1): 105-32; Parker J M, Guo D, Hodges R S, Biochemistry 1986 Sep.23; 25(19): 5425-32; Welling G W, Weijer W J, van der Zee R,Welling-Wester S; FEBS Lett. 1985 Sep. 2; 188(2):215-8]. In oneembodiment the peptide is determined via the metaepitopicity subprogramfrom Metalife AG (Metatope™, Metapark, 79297 Winden, Germany).

In one embodiment, the viral capsomer is derived from a first virus andthe peptide is derived from a second virus which is not the same as thefirst virus.

In one embodiment, the peptide is derived or can be obtained from avirus selected from the group of non-enveloped viruses, comprisingPapovaviridae, in particular polyoma and papilloma viruses,Iridoviridae, Adenoviridae, Parvoviridae, Picornaviridae, in particularpolio viruses, Caliciviridae, Reoviridae and Birnaviridae.

In one embodiment, the peptide is derived or can be obtained from avirus selected from the group of enveloped viruses, comprisingPoxviridae, Herpesviridae, Hepadnaviridae, Retroviridae,Paramyxoviridae, Sendaiviridae, Orthomyxoviridae, Bunyaviridae,Arenaviridae, Toroviridae, Togaviridae, Flaviviridae, Rhabdoviridae andFiloviridae.

In one embodiment, the peptide does not comprise any peptide epitopewhich is typically used for recording the infection status, diseasestatus or vaccination status, for example in the course of vaccinationprograms.

An example of such a peptide epitope used for recording the infectionstatus is the peptide epitopes of the non-structural protein (NSP)“3ABC”. This is a protein of the foot-and-mouth virus, which is not partof the viral envelope but to which an animal infected with the livingvirus produces antibodies regardless (in addition to the antibodies tothe structural, i.e. envelope, proteins). In modern vaccines, however,such non-structural proteins of the foot-and-mouth disease virus havebeen removed so that an animal vaccinated with a vaccine prepared inthis way does not produce any antibodies to the “3ABC” epitopes. If aninfected animal or a vaccinated animal is tested for antibodies to“3ABC” epitopes by means of an immunochemical assay (for example ELISA),then the infected animal shows a positive response and the vaccinatedanimal shows a negative response. Consequently, “3ABC” or the epitopespresent therein are used as differentiation markers when recording theinfection status or vaccination status of animals. This enablesvaccinated and infected animals to be distinguished from one another. Anassay of this kind for “3ABC” is available from Intervet(http://www.intervet.com).

Preferably, the peptide does not derive or cannot be obtained from anagent, for example a virus, bacterium or a eukaryotic cell, which entersthe organism of the living being in the form of a vaccine or medicamentor via the food chain or, under normal conditions of life of said livingbeing, via the environment and/or to which antibodies are produced insaid living being under normal conditions of life, it being preferredthat the peptide does not derive or cannot be obtained from a virusselected from the group comprising CSF virus (swine fever virus),foot-and-mouth disease virus, PPV (porcine parvovirus), influenza virus,in particular influenza A virus, bovine leukemia virus (EBL virus),bovine herpes virus (BHV1), bovine viral diarrhea virus (MD virus),bovine polyoma virus (BpyV), rotavirus, porcine herpes virus 1,pseudorabies virus, PRRS virus and TGE virus.

In one embodiment, the peptide does not derive from Leptospira, inparticular L. grippotyphusa, L. tarassovi, L. canicola, L. pomona, L.bratislava, Chlamydia, in particular C. psittaci, Brucella, inparticular B. abortus, B. canis, B. melitensis, Mycobacterium, inparticular M. avium subsp. paratuberculosis or Coxiella, in particularC. burnetii.

In one embodiment, the peptide is an artificial peptide.

“Artificial” in this connection means that the peptide has a sequencewhich is of artificial origin, i.e. is a “fantasy sequence”. However,this should not rule out the possibility of finding in a database suchan artificial sequence belonging to an organism. The only singlecriterion of such a sequence is the fact that it has been selectedwithout taking into account or knowing its presence in a database.

In one embodiment, the at least one peptide has been coexpressed withthe capsomer protein, starting from a DNA encoding said at least onepeptide and said capsomer protein.

In one embodiment, the peptide is linked to the viral capsomer via astructure which mediates interaction, with the interaction-mediatingstructure preferably being located on the viral capsomer.

The interaction is preferably a hydrophobic interaction, a covalentbond, an ionic bond or a hydrogen bond between the viral capsomer andthe at least one peptide.

In one embodiment, the structure which mediates interaction haspreferably at least one bifunctional crosslinker, which is preferably aheterobifunctional crosslinker which particularly preferably has amoiety which is reactive to amino groups and a moiety which is differenttherefrom and which is reactive to sulfhydryl groups.

In one embodiment, the bifunctional crosslinker is selected from thegroup comprising maleimide derivatives, alkyl halides, aryl halides,isocyanates, glutardialdehydes, acrylating reagents and imido esters.

In one embodiment, the structure which mediates interaction has at leastone affinity-increasing group which preferably is selected from thegroup comprising 4-iodoacetamidosalicylic acid, p-arsonic acidphenyldiazonium fluoroborate and derivatives thereof.

The viral capsomer is preferably associated with two or more peptides asdefined above.

In this connection, the two or more peptides may have the same sequenceor a different sequence. In the case of more than two peptides, thesemay have the same sequence or one or more different sequences.

In one embodiment, the viral capsomer and/or the at least one peptideare in the form of the nucleic acid coding therefor.

In one embodiment, the composition furthermore comprises an adjuvant.Preferably, the adjuvant is selected from the group comprising MontanideIMS 1312® and Quillaja Saponin (QuilA). Also suitable are CpG-DNA,aluminum adjuvants (e.g. aluminum hydroxide gels such as Alhydrogel),other saponins, aqueous adjuvants (such as, for example, Montanide IMS1313®, Montanide IMS 1314®), water/oil emulsions, oil/water emulsions,water/oil/water emulsions (with metabolizable oils or withnonmetabolizable mineral oils), ISCOMs, liposomes, LPS and derivatives(such as, for example, MPL=Monophosphoryl Lipid A) as adjuvants.

Preferably, upon singular administration of said composition to a livingbeing, the viral capsomer elicits in said living being an immuneresponse which can still be detected at least 18 weeks, preferably atleast 20 weeks, more preferably at least 24 weeks, post administration.

Preferably, the immune response manifests itself in the form of anincreased anti-viral capsomer-IgG and/or -IgA titer and/or an increasedanti-viral capsomer protein-IgG and/or -IgA titer and/or an increasedanti-peptide-IgG and/or -IgA titer, it being preferred that theincreased anti-viral capsomer/viral capsomer protein/peptide-IgG and/or-IgA titer is at least 1:64, more preferably at least 1:128, which, inone embodiment, is also still detectable at least 18 weeks, preferablyat least 20 weeks, more preferably at least 24 weeks, postadministration.

Detection is preferably carried out by means of an enzyme-immunologicalor immunochemical accelerated test or ELISA, which, in one embodiment,is performed on a body fluid selected from the group comprising meatjuice, blood, whole blood, serum, plasma, lymph, urine, saliva, milk andsemen.

The objects of the present invention are likewise achieved by a methodof labeling agents administered to living beings, characterized by thefollowing steps:

a) preparing a composition of the invention, as defined above, by addinga protein complex or viral capsomer as defined above to an agent to belabeled, as defined above,

b) administering said composition to a living being,

c) detecting the immunoresponse caused by said administration in saidliving being by means of an enzyme-immunological or immunochemicalmethod.

Preference is given to the immune response comprising a formation ofantibodies, said antibodies preferably being secreted antibodies and/orantibodies exposed on lymphocyte surfaces.

Preferably, detection takes place in a body fluid selected from thegroup comprising meat juice, blood, whole blood, plasma, lymph, serum,saliva, milk, urine and semen.

In one embodiment, the lymphocytes are B-lymphocytes and/orB-lymphocytes in combination with T-lymphocytes.

Preferably, the administration to a living being is carried out once orseveral times, in the latter case at intervals of several weeks,preferably 1-4 weeks.

In one embodiment, the agent is a medicament, a vaccine or stored blood,preferably an anti-infectious agent, in particular an antibiotic.

The objects of the present are also achieved by using the method of theinvention and/or the composition of the invention for labeling livingbeings, said living being preferably being a non-human mammal, morepreferably a mammal selected from the group comprising cattle, pigs,sheep, horses, hares, rabbits, dogs, cats, llamas, camels, marinemammals such as ceteaceans, seals and harbor seals.

The objects of the present invention are also achieved by using themethod of the invention and/or the composition of the invention forimmunological monitoring, it being preferred to check living beings orpopulations of living beings, as to whether they have come into contactwith a particular agent, for example a vaccine, a medicament, afoodstuff, etc.

The objects of the present invention are also achieved by an antibodydirected against the viral capsomer and/or against the at least onepeptide of the composition of the invention.

The objects of the present invention are also achieved by an antibodydirected against the aforementioned antibody.

The objects of the present invention are also achieved by an acceleratedtest comprising the last-mentioned antibody and/or the viral capsomer asdefined above and/or the viral capsomer as described above inassociation with the at least one peptide as defined above.

Preference is given to the antibody and/or the viral capsomer beingcoupled to a reporter reagent.

Examples of the reporter reagent are colloidal gold, fluorescent dyes,biotin, alkaline phosphatase or peroxidase, preferably horseradishperoxidase. More preferably, the reporter reagent is colloidal gold.

The term “virus-like particle” (VLP), as used herein, refers to anagglomerate of viral proteins, which is incapable of replicating withthe aid of the host cell metabolism, but which has the phenotype of aviral envelope, for example under an electron microscope. A virus-likeparticle, as used herein, represents a noninfectious viral envelope orparts thereof. The term “virus-like particle”, as used herein, is usedsynonymously with “capsoid”. The term “virus-like particle”, as usedherein, is to be distinguished from the individual building blocks of aviral envelope, the “viral capsomers”. Thus, numerous viral envelopesconsist of subunits, called capsomers, which make up the envelope. These“capsomers” in turn usually consist of one or more proteins, calledviral capsomer proteins. The term “viral capsomer protein”, as usedherein, refers to a subunit of a viral capsomer, it being possible forsaid viral capsomer to be composed of one or more viral capsomer proteinmolecules of one or more types.

The use “viral capsomer in association with other viral capsomers”, asused herein, refers to a combination of a variety of viral capsomers,for example of two or more viral capsomers. The term preferably refersto the combination of at least two viral capsomers. The term“association with other viral capsomers” may also include a completeviral capsoid. However, preference is given to an “association withother viral capsomers” not being a complete viral capsoid. The term“pentamer”, “hexamer” or “heptamer” when used in order to describe aviral capsomer in more detail, refer to a combination of five, six orsevel viral capsomer proteins, each of which result in a viral capsomer.The term “viral capsomer”, as used herein, is to be understood asmeaning that the viral capsomer is not present in the form of a capsoid(or synonym: viral capsoid).

The use of viral capsomers in a composition to be administered has beenshown to result in immune titers of antibodies/B-cells, which lastdistinctly longer or are distinctly higher than those which can beachieved by the immunogens used in the prior art (e.g. KLH, GFP, or elsewhole virus-like particles). The viral capsomers of the invention can beprepared recombinantly in a simple manner and exhibit no undesired sideeffects whatsoever in the organism to which the composition isadministered.

In contrast to the use of complete virus-like particles, the immuneresponse achieved when using viral capsomers is equally high or higher,without the complicated reconstitution step for preparing the viralcapsoids or VLPs. As a result, the method of the invention isconsiderably less expensive. Moreover, in the case of the viralcapsomers of the invention, an undesired crossreactivity can be ruledout, enabling viral capsomers only then to be used for labeling ofagents to be administered or in a labeling process. The viral capsomersof the invention are soluble in aqueous solution and are therefore alsosuitable for the use in living animals, in particular farm animals.

In one embodiment of the present invention, the viral capsomers ofmurine polyoma virus have proved particularly advantageous. Murinepolyoma virus has been linked with tumorigenesis in rodents. There areother species-specific or family-specific polyoma viruses, at least someof which have been found also to be tumorigenic, for example the primatevirus SV40, bovine polyoma virus (BpyV), two human polyoma species (JCand BK), the two latter having been linked with progressive multifocalleukoencephalopathy (PML) and ureter stenosis in humans. Murine polyomaviruses is a double-stranded DNA virus belonging to the Papovaviridaefamily. The double-stranded DNA molecule consists of approximately 5000bp and encodes five transcripts (T, t=early proteins, VP1, VP2 andVP3=late structural proteins). The viral envelope consists of threeenvelope proteins, VP1, VP2 and VP3, which may be used for the formationof virus-like particles (VLPs). However, the formation of VLPs does notrequire the presence of all three proteins. The isolated major envelopeprotein VP1 has been shown to form VLPs under particular conditions tobe set by the person carrying out the experiment. The infectious murinepolyoma virus has an envelope whose structure is formed by two shells,the outer shell of which consisting exclusively of VP1 and the innershell of VP2 and VP3. It is therefore possible to generate noninfectiousempty shells (virus-like particles) which consist exclusively of VP1.These empty envelopes may be assembled in vitro, for example usingrecombinant VP1, and are referred to, as used herein too, as “capsoids”,“viral capsoids” or “VLPs” or “virus-like particles” if they form acomplete envelope. The VLPs are approximately 50 nm in diameter (asdetermined by means of electron microscopy) and are formed by 360 VP1molecules which are arranged in 72 pentamers. Depending on theassembling conditions, however, it is also possible for smaller capsoidsof 26 nm or 32 nm to be formed (Salunke et al., 1989, Biophys. J. 56(5): 997-990). A VP1 pentamer is a “viral capsomer”, i.e. acapsoid-forming subunit. In this special case, a viral capsomer consistsof five viral capsomer proteins, i.e. five VP1 molecules. The formationof pentamers (i.e. capsomer formation) is a process of spontaneousself-assembly which, in the case of recombinant expression of a VP1protein in a host cell, for example E. coli, takes place immediatelyafter expression, i.e. in vivo.

Reference is now made to the figures in which:

FIG. 1 depicts both a sequence comparison of the VP1 protein sequencesof mouse polyoma virus (strain PG) and of hamster polyoma virus andpossible sites of integration in outer loop regions of wild type VP1(mouse polyoma virus),

FIG. 2 depicts a VP1 expression vector (pET-9a/VP1),

FIG. 3 depicts the VP1 protein-encoding DNA sequence of the mousepolyoma virus strain BG,

FIG. 4 depicts the integration of a foreign epitope into wild type VP1by means of a PCR-based site-directed mutagenesis,

FIG. 5 depicts the purification of VP1-peptide capsomers, with FIG. 5Adepicting a preparative gel filtration of the marker vaccine VP1-BC2,FIG. 5B depicting an SDS-PAGE analysis of the corresponding VP1fraction, and FIG. 5C depicting a PCS (Photon Correlation Spectrometry)analysis of the corresponding VP1 fraction,

FIG. 6 depicts the assembly of VP1 capsomers to capsoids, with FIG. 6Adepicting a gel filtration profile, FIG. 6B depicting the correspondingPCS measurement, FIG. 6C depicting the results of an analytical gelfiltration and FIG. 6B depicting the results of a PCS measurement,

FIG. 7 depicts the time course of the immune responses in pigs, measuredas anti-VP1 titer after immunization,

FIG. 8 depicts the time course of the immune responses in pigs, measuredas anti-peptide titer after immunization,

FIG. 9 depicts the time course of the immune responses in pigs afterimmunization with VP1 pentamers with and without boost immunization,measured as anti-VP1 titer over a period of 20 weeks,

FIG. 10 depicts the time course of the immune responses in cattle to animmunization with different doses of viral capsoids over a period of 20weeks,

FIG. 11 depicts the time course of the immune responses in cattle to animmunization with VP1 capsoids and VP1 pentamers over a period of 24weeks, and

FIG. 12 depicts the diagrammatic structure of an embodiment of acorresponding accelerated test (12A) and a photographic view (12B) of anembodiment of such an accelerated test.

Reference is now made to the examples which are presented herein for thepurpose of illustration and not by way of limitation.

EXAMPLE OF VIRAL CAPSOMERS AS LABELING VACCINE

In this embodiment, soluble viral capsomers associated with a peptidesequence were used for labeling. The viral capsomers in this exampleconsisted of pentamers of the murine polyoma virus envelope protein VP1.In addition, murine VP1 viral capsomers were employed in order to beable to compare the immune responses of capsomer and capsoid. The VP1viral capsoids consist in each case of 72 capsomers (pentamers).

The peptide was selected according to the abovementioned definitions. Inthis embodiment, VP1 was linked to the peptide by directly cloning thepeptide sequence into VP1. Piglets were selected as use examples. Theimmune response manifested itself here in the form of increased anti-VP1and peptide-IgG titers, the antibodies were detected in the blood serumby means of ELISA.

Example 1 Binding of the Peptide to the Capsomer

In this embodiment, binding was effected by inserting the peptidesequence directly into murine VP1 polyoma virus.

For this purpose, surface-exposed, flexible regions within the VP1structure were selected. The selection was carried out on the basis ofthe X-ray crystal structure of murine polyoma VP1 as pentamericasssemblage (1SID, PDB; according to Stehle and Harrison, 1996,Structure 4(2):183-94). The structural analysis with respect to thesecondary, tertiary and quaternary structures was carried out with theaid of the VMD program (Virtual Molecular Dynamics) V.1.7.2 (TheoreticalBiophysics Group, University of Illinois and Beckman Institute). Inaddition, the biochemical parameters: polarity, hydrophobicity and ionicinteractions, were taken into account. Furthermore, insertion of thepeptide should not influence the formation of pentamers.

On the basis of these criteria, the following regions (FIG. 1) wereselected:

-   -   BC2 loop (sequence position 80-88)    -   HI loop (sequence position 291-296)    -   FG loop (sequence position 246-249)

(The loop regions were named according to Stehle and Harrison, 1996)

An extension of the strategy is the possibility of not only carrying outone peptide integration per VP1 monomer but also integratingsimultaneously in different loop regions

a. the same epitope, in order to thereby increase the immunogen dose andthus the immune response;

b. various epitope sequences, in order to thereby modulate, different,specific immune responses.

The embodiment described below is the insertion of the peptide into theBC2 loop.

The study by Gedvilaite et al., 2000 (Virology 273(1): 21-35) involvedinserting an epitope of hepatitis B virus into various regions ofhamster polyoma VP1. However, since the sequence identity of hamsterpolyoma VP1 and murine polyoma VP1 is only 63.6% (FIG. 1), a directcomparison with this study is not possible.

FIG. 1 depicts, highlighted by a gray box, the core regions of the outerloop regions in wild type VP1, which were utilized for the integrationof foreign sequences; moreover, the figure depicts a homology comparisonof the VP1 protein sequences of mouse polyoma virus strain BG (in eachcase line 1) and hamster polyoma virus (in each case line 2).

Example 2 Cloning of the VP1 Mutants

Depending on the primary structure or length (8-20 amino acids) of theforeign epitope to be inserted and on the particular VP1 integrationsite, the loop regions of the wild type sequence were adjusted by“compensating deletions” of varying length (Δ=0 to 12 amino acids). Onthe one hand, it was intended to avoid disadvantageous structuralalterations of the VP1 protein with effects on the solubility andassembly ability. On the other hand, it was intended to ensure goodepitope exposition on the surface.

This adjustment was carried out with the aid of the 3D crystal structureimage of wild type VP1 (Stehle and Harrison, 1996, EMBO J. 16(16):5139-48 and Stehle and Harrison 1996, Structure 4(2): 165-82) and bymeans of suitable algorithms and structural predictions, using theProtean™ program (DNA-STAR Inc., Madison (Wis.), USA), according to:

-   -   Surface Probability—Emini et al., 1985, J. Virology 55: 836-9    -   Flexibility—Karplus and Schultz, 1965, Naturwissenschaften 72:        212-3    -   Hydropathy—Kyte and Doolittle, 1982, J. Mol. Biol. 157: 105-32    -   Hydropathy and Antigenicity—Hopp and Woods, 1981, Proc. Natl.        Acad. Sci. 78: 3824-8    -   Antigenicity—Jameson and Wolf, 1968, CAPIOS 4: 181-6

In order to be better able to achieve the abovementioned criteria, it ispossible to generate from the abovementioned types of integrationfurther variants in which the peptide is inserted in the particular loopregion with flanking, symmetrically arranged linkers. Said linker may beby way of example a tetrapeptide-serine/glycine linker ( . . .Ser-Gly-Ser-Gly-peptide-Gly-Ser-Gly-Ser . . . ). This peptide linkerincreases firstly the flexibility and secondly the hydrophobicity at theintegration site. Other linkers having similar properties, such asserine/glycine dipeptide linkers, serine/glycine hexapeptide linkers,etc. and polyglycine linkers, are also conceivable for this purpose(Imanishi et al., 2000, Biochemistry 39(15): 4383-90 and Arakaki et al.,2002, Protein Expr Purif 25(2): 241-7).

2.1 Generation of the Expression Mutants

The integrations of the peptide-encoding foreign sequences and thedeletions of the wild type sequences were generated by a PCR-basedsite-directe mutagenesis reaction directly with the entire, circular VP1expression vector pET-9a/VP1 (5526 bp) (FIG. 2) as template. Thisrendered further subclonings unnecessary. The VP1 DNA (1155 bp)contained in the vector pET-9a (Novagen) is derived from the murinepolyoma virus strain BG (GenBank/NCBI: accession number AF442959, FIG.3) and is expressed as a protein of 384 amino acids without fusion tagunder the control of a T7 promoter.

The VP1 expression vector pET-9a/VP1 depicted in FIG. 2 has thefollowing molecular properties which can be found in the followingtable: TABLE 1 Molecular properties: Name Start End Description Kann 819 7 C Kanamycin resistance gene pBR322 ori 1711 865 C pBR322 origin ofreplication pT7 3711 3727 T7 promoter VP1 3791 4945 VP1-DNA tT7 50775123 T7 terminator

The hybrid primer pairs used for mutagenesis contained at their 3′ endsadjacent wild type sequences which were complementary to the sense andantisense strands of the target DNA. In the case of deletions, theoligonucleotides hybridized with the wild type sequence at a greaterdistance from one another and thus left out a defined region.

The DNA sequence encoding the foreign epitope was distributed to the 5′ends of the two hybrid primers and contained optimized codons foroverexpression in E. coli (Shaun D. Black, University of Texas HealthCenter at Tyler; http://psyche.uthct.edu/shaun/SBlack/codonuse.html).The 5′ terminal positions were designed so as to contain in each case aportion of a unique recognition sequence for a restriction endonuclease(FIG. 4).

2.2. Example of a Mutagenesis in the Region Encoding the BC2 Loop

Unless stated otherwise, standard methods, for example according toSambrook and Russell (Molecular Cloning, 5th ed., Cold Spring Harbor(N.Y.): Cold Spring Harbor Laboratory Press; c2001) were used.

The PCR mutagenesis was carried out using the oligonucleotides 080BN093f and 080BN093 r (FIG. 4) and 100 ng of circular pET-9a/VP1 template DNAin 50 μl reaction mixtures according to standard methods. To reduce theerror rate, PfuTurbo®DNA polymerase (Stratagene) with proofreadingactivity was used, only PAGE-purified oligonucleotides were utilized andthe number of PCR cycles was limited to from 10 to no more than 18cycles. The elongation was carried out at 68° C. for 2 minutes each per1 kb. The PCR was carried out according to the following cycle profile:

Part of the reaction mix was used for analyzing the linear vectorproduct produced with respect to quantity and quality, in an agarosegel.

The remaining reaction mixture was incubated with 20 U of DpnIrestriction endonuclease (New Enland Biolabs) at 37° C. for 1 h, wherebythe methylated pET-9a/VP1 template DNA was selectively degraded.

After a “final polishing” with 1 U of PfuTurbo®DNA polymerase(Stratagene) at 68° C. for 0.5 h, in order to generate a complete set ofblunt ends, the PCR products were isolated according to theE.Z.N.A.®Cycle-Pure protocol (PeqLab). The 5′ ends were phosphorylatedby incubation with 5 U of T4 polynucleotide kinase (New England Biolabs)at 37° C. in a ligation buffer compatible therewith (New EnglandBiolabs) for 0.5 h.

The linear amplification products were circularized by a 2 h shortligation at room temperature, using 400 NEB units (˜6 Weiss units) of T4DNA ligase (New England Biolabs), and this reaction mixture wassubsequently used for transforming the E. coli strain XL1 Blue (recA1endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F′proAB lacIqZDM15 Tn10(Tet^(r))]; Stratagene).

After selection on LB kanamycin agar, individual clones were amplifiedand the mini-prepared plasmid DNA (QIA-prep®Spin Miniprep Kit, Qiagen)was analyzed by restriction digest, in this case by MscI endonuclease incombination with NdeI endonuclease (New England Biolabs), with respectto the recombinant portion and the correct blunt-end fusion site.Recombinant plasmids were furthermore analyzed with regard tocompleteness of the plasmid by EcoNI restriction (3 cleavage sites inthe vector sequence).

After fulfilling the two restriction criteria, the DNA of theVP1-encoding region was sequenced according to Sanger (Proc. Natl. Acad.Sci. USA (1977) 74, 5463-67) using the “ABI PRISM® Big Dye Terminators v2.0 Cycle Sequencing Kit” (Applied Biosystems). In the case of anerror-free sequence, the recombinant construct was transformed forexpression into the E. coli strain BL21 (DE3) (F⁻ ompT hsdS_(B)(r_(B)⁻m_(B) ⁻) gal dcm (DE3); Novagen).

FIG. 4 depicts the diagrammatic representation of the integration of aforeign epitope (12 amino acids) with a compensating deletion (12 aminoacids) in the BC2 loop of wild type VP1 by a PCR-based site-directedmutagenesis.

Example 3 Preparation of Recombinant VP1-peptide Capsomers

In this embodiment, the VP1-peptide capsomer, referred to as VP1-BC2hereinbelow, was purified recombinantly from E. coli.

When expressed in E.coli, the VP1 protein is in the form of pentamers(=capsomers). The assembly to capsomers must be induced, afterpurification of the pentamers, specifically by adding high saltconcentrations (e.g. (NH₄)₂SO₄), oxidizing agents and Ca²⁺. In contrast,aggregates may form spontaneously depending on the purification andstorage conditions of the capsomers. These aggregates are unspecificassemblages of at least two capsomers. The ratio of monomeric capsomersto capsomer aggregates and the size of the aggregates formed depend onthe external conditions (see below). In this connection, the aggregatescan be distinguished clearly from the capsoids: they do not form anenvelope surrounding a cavity, but unspecific, irregular shapes.Aggregates may be the result of:

-   -   VP1-pentamer concentrations ≧1 mg/ml    -   vigorous or long-lasting shaking, vortexing, etc.    -   working at room temperature    -   long-term storage of the pentamers at temperatures ≧0° C.    -   no addition of reducing agents (e.g. DTT) and/or        oxidation-inhibiting reagents (EDTA) to the buffer.

Depending on the buffer conditions, movements, temperature and type andduration of storage, the proportion of aggregates in the pentamersolution may be from 0 to above 50%. The size may also greatly vary,depending on the conditions (from approx. 2 to more than 20 pentamers).However, it is not possible to give an exact size, since the sizedepends on the external conditions, as listed above.

The aggregates may be detected by:

-   -   PCS measurement: While single VP1 capsomers have a specific size        of 8-10 nm and capsoids have a size of 26 nm, 32 nm or 45 nm,        aggregates have an unspecific size distribution of 20-100 nm, in        extreme cases even more than 100 nm.    -   Gel filtration: Aggregates elute earlier than VP1 pentamers.        Therefore, two separate signals (capsomer aggregates and        monomeric capsomers) can be recognized in the gel filtration        profile of VP1 pentamers in the case of aggregate formation. The        proportion of aggregates can be quantified by gel filtration.    -   Electron microscopy: VP1 capsoids can clearly be recognized as        envelopes containing a cavity, constructed from VP1 capsomers.        In contrast, the aggregates can be seen as structureless,        irregular capsomer assemblage. However, quantification of the        aggregates (proportion/size) by electron microscopy is not        possible.

The following example describes the protocol of the purification ofVP1-BC2; the VP1 wild type was isolated according to the same principle.

After transformation of the VP1-BC2 plasmid into the E.coli strainBL21(DE3), expression, cell harvest and lysis, the protein was found tobe in the soluble supernatant. Further isolation of the capsomers wascarried out tag-free by a three-stage process.

After cation and anion exchanger, after-purification was carried out byway of a preparative gel filtration (HiLoad 16/60 Superdex 200 prepgrade, Amersham Pharmacia) in PBS buffer. The gel filtration run in FIG.5A shows only one peak at 55-65 ml. The SDS gel of the correspondingfraction shows VP1-BC2 (calculated mass=44 kDa) (FIG. 5B). Evaluationusing the gel documentation system “ImageScanner” (Pharmacia) and thesoftware “ImageMaster 1D, Version 4.00” (Pharmacia) reveals a purity of75%. Checking the eluted VP1 fraction by photon correlation spectrometry(PCS) measurement using a high performance particle sizer (ALV-Sizer2.9, ALV-NIBS) shows a particle size distribution of around 8 nm (FIG.5C), which approximately corresponds to the size of 8-10 nm for VP1wild-type capsomers, calculated by electron microscopy. Since neitherthe gel filtration profile nor the PCS measurement revealed additionalprotein or particle fractions, assembled capsoids or aggregates ofcapsomers may be present only to a small extent at this point in time.

After sterile filtration, the capsomers were used for labeling.

FIG. 5A depicts a preparative gel filtration of the marker vaccineVP1-BC2 over a HiLoad 16/60 Superdex 200 column. Only one VP1 fractionis detected. FIG. 5B depicts an SDS-PAGE analysis of the correspondingVP1 fraction. M=marker; VP=VP1 pentamer. FIG. 5C depicts the PCSanalysis of the corresponding VP1 fraction.

Example 4 Assembly of the VP1 Capsomers

The assembly requires an aggregate-free capsomer fraction. In this case,the 3rd purification stage was carried out by way of preparative gelfiltration (HiLoad 26/60 Superdex 200 prep grade, Amersham Pharmacia) inKB1 buffer (50 mM Na₂HPO₄, 150 mM NaCl, 2 mM EDTA, 5% glycerol, pH 6.8).

Here too (FIG. 6A), the gel filtration profile has only one VP1 peak(elution at 130-150 ml). Checking by PCS (photon correlationspectrometry) measurement indicates VP1 particles of around 10 nm insize (see FIG. 6B), corresponding to the abovementioned size ofpentamers.

FIG. 6A depicts a preparative gel filtration of VP1-EC2 over a HiLoad26/60 Superdex 200 column. Only one VP1 fraction is detected. FIG. 6Bdepicts the results of a PCS measurement of the correspponding VP1fraction. FIG. 6C depicts the analytical gel filtration over a TSK Gel G6000, PWXL column after assembly of the VP1 capsomers. The main elutionfraction shows capsoids. FIG. 6D depicts a PCS analysis after assemblyof the VP1 capsomers.

Subsequently, the capsomers were assembled to capsoids in an additionalstep according to standard methods (Stehle et al., 1994, Nature 369(6476): 160-3 and Stehle et al., 1996, Structure 4(2): 165-82), whichare known to the skilled worker. After the final dialysis, VP1-BC2 wasin PBS+0.7 nM CaCl₂.

The size of average VP1 capsoids is indicated to be 45 nm, but smallercapsoids of 32 nm or 26 nm in size may also be produced, depending onthe assembly conditions (Salunke et al., 1989, Biophys J. 56(5):887-900). The PCS measurement carried out here following assemblyindicated a particle size distribution of around 30-40 nm (FIG. 6D),approximately corresponding to the abovementioned data for capsoids. Inaddition, the assembly was checked by gel filtration using a TSK Gel G6000-PWXL column (Toso Haas) (FIG. 6C). While the main elution fractionat 8 ml contains capsoids, non-assembled pentamers were present only ina smaller side fraction at 11 ml.

The VP1-BC2 capsoids were used for labeling, after sterile filtration.

Example 5 Analytical Testing of the Purified VP1 Mutants

5.1. Purity Analysis of the Protein by HPLC/mass Spectroscopy

HPLC/mass Spectroscopy

Besides SDS-gel analysis, the purity and exact mass were checked in thisexample via LC ESI-MS. The determination was carried out by means of theAgilent LC/MS 1100 Series and the “Agilent ChemStation, Version 08.03”software.

The VP1-BC2 mutant was, after reduction with DTT (dithiothreitol)fractionated by reversed phase HPLC with a gradient of 0-90%acetonitrile in H₂O containing 0.1% trifluoroacetic acid over a PLRP-S,300 Å, 150×4.6 mm (Polymerlabs), and the protein was detected bymeasuring the absorption at 214 nm and 280 nm. The mass was determineddirectly thereafter by ESI-MS (electrospray ionization massspectroscopy).

calculated mass of VP1-BC2: 44 334.2 Da. found mass of VP1-BC2: mainmass: 44 316.6 Da (−17.4 Da).

In addition, an additional mass of 47 717.5 Da (+3401 Da) with aproportion of approx. 20% was found, which might indicate contaminationswith E.coli proteins.

The data were recorded with an uncertainty interval of 12.39 Da and astandard deviation of 4.67 Da.

5.2. LPS Determination

This example utilized the LAL test (LAL=Limulus Amebocyte Lysate) byCharles River (Charleston, USA) for the determination of endotoxins.This test is based on the reaction of the LAL reagent with endotoxins,which proceeds with clouding and gel formation. The test was carried outaccording to the manufacturer's information following the kineticturbidity method. The turbidity rate was measured by means of an ELISAreader (B Elx808 BIO-TEK reader) and evaluated using the “EndoScan VSoftware” (Charles River).

The result for the example of the VP1 mutant VP1-BC2 used here was anendotoxin concentration of:

185 EU/mg for VP1-BC2 pentamers

<42 EU/mg for VP1-BC2 capsoids

Example 6 Preparation of the Vaccine Doses for Pigs and Sterile Controls

In this example, in each case 50 μg or 200 μg of VP1-BC2 per vaccinedose were aliquoted under sterile conditions, admixed with sterilephysiological saline (0.9% NaCl) to a volume of 1 ml and admixed with 1ml of the adjuvant Montanide® IMS 1312 (Seppic, France). The finishedvaccine solution was incubated with rolling at 4° C. overnight. Sincethe incubation was carried out with agitation and without addition ofreducing agents, the capsomers could possibly have formed aggregatesconsisting of several pentamers, but this should not be regarded asfact. In contrast, the capsoids are extremely stable and cannot form anyfurther aggregate forms.

For the sterile control, in each case 100 μl of the VP1 solution wereremoved, streaked onto CASO (Merck) agar plates and incubated at 37° C.After 24 h at 37° C., no colonies were found.

The vaccine doses for cattle were prepared analogously, but the adjuvantused was Quil A (1 mg/ml saline, Superfos, Denmark).

Example 7 Vaccination Schedule and Immunization

7.1. Vaccination Schedule

Pigs:

As an example, the immune response to the recombinant VP1-BC2 proteinwas first checked using piglets. The following problems were examined:

-   -   immune response to VP1-BC2 capsoids or pentamers    -   immune response to 50 μg or 200 μg of VP1-BC2

16 piglets (male and female) aged 19-21 days were selected and dividedinto 4 groups: Group Vaccine dose Adjuvant Group 1 (4 animals): 200 μgof VP1-BC2, pentamer IMS 1312 Group 2 (4 animals):  50 μg of VP1-BC2,pentamer IMS 1312 Group 3 (4 animals): 200 μg of VP1-BC2, capsoid IMS1312 Group 4 (4 animals):  50 μg of VP1-BC2, capsoid IMS 1312

Cattle:

For dose comparison (capsoids 10 μg/dose or 100 μg/dose), in each case 5cattle per dose were vaccinated; for a pentamer/capsoid comparison, ineach case 2 cattle were vaccinated.

7.2. Immunization and Taking Blood

Pigs: The vaccination was carried out intramuscularly at the base of theear. No group was boosted. Blood samples were taken from the vena cavacranialis on day 0 (preimmune serum), 14, 28 and 42.

Cattle: The vaccination was carried out subcutaneously on the side ofthe neck. The blood was taken from the external jugular vein on the dayof immunization (preimmune sera) and after 2, 4, 6, 8, 12, 16, 20 weeks(dose comparison) or 2, 4, 8, 12, 16, 20, 24 weeks (pentamers/capsoidscomparison).

Example 8 Detection of the Immune Response

In this example, the immune response manifested itself in the form of anincreased anti-VP1 and anti-peptide-IgG titer. The titers weredetermined here by ELISA-testing the blood serum.

Pigs:

To detect the anti-peptide or -VP1 titers, the wells of an ELISA plate(C-MaxiSorp, Nunc) were coated with synthetic peptides of thecorresponding sequence or with wild type-VP1 capsomers or correspondingcapsoids. Incubation of the corresponding solutions at 4° C. overnightwas followed by washing with PBS-T (PBS+0.5% Tween 20). Blocking wascarried out with 1% BSA (bovine serum albumin, fraction V, Roth) in PBS,excess BSA was removed by washing with PBS-T. Subsequently, 100 μl oftest serum, diluted in PBS-T with 0.5% BSA, negative and positivecontrols, were applied. The test serum was tested in decreasingconcentration steps, with the serum concentration halved in each step(dilution 1:4; 1:8; 1:16 etc.). The samples were incubated on the platesfor 1 h, excess reagent was removed by washing with PBS-T andaffinity-purified biotin-coupled goat anti-pig IgG (Dianova) was added.After 3 more washing steps with PBS-T, the biotin-conjugated antibodywas labeled by incubation with streptavidin-peroxidase (Roche). Excessreagent was removed with PBS-T. Development was carried out using thesubstrate 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)diammonium salt (Roche) which reacts with peroxidase to give a greencolor. The reaction was stopped by adding oxalic acid. The quantitativeevaluation was carried out by means of an ELISA reader (MultiscanAscent, Labsystems) by determining the optical density at 405 nm with492 nm as reference (OD_(405/492 nm)).

Cattle:

To detect the anti-VP1 titers, the wells of an ELISA plate (C-MaxiSorp,Nunc) were coated with wild type-VP1 capsomers or correspondingcapsoids. Incubation of the corresponding solutions at 4° C. overnightwas followed by washing with PBS. Blocking was carried out with 1% BSA(bovine serum albumin, fraction V, Roth) in PBS, excess BSA was removedby washing with PBS-T (PBS+0.05% Tween 20). Subsequently 50 μl of testserum, diluted in PBS-T with 50 mM EDTA, negative and positive controls,were applied. The test serum was tested in decreasing concentrationsteps, with the serum concentration being halved in each step (dilution1:4; 1:8; 1:16 etc.). The samples were incubated on the plates for 1 h,excess reagent was removed by washing with PBS-T and affinity-purifiedgoat anti-bovine IgG peroxidase conjugate (Dianova) was added. Excessreagent was removed by PBS-T. The development was carried out using thesubstrate 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)diammonium salt (Roche) which reacts with the peroxidase to give a greencolor. The reaction was stopped by adding oxalic acid. The quantitativeevaluation was carried out by means of an ELISA reader (MultiscanAscent, Labsystems) by determining the optical density at 405 nm, with492 nm as reference (OD_(405/492 nm)).

To calculate the antibody titers, first the cut-off value was determinedas follows:

cut-off=average of OD_(405/492 nm) of the negative control+3*standarddeviation.

A dilution of the test serum is denoted positive, if the OD_(405/492 nm)is higher than the cut-off value. The antibody titers are obtained bycalculating the log 2 of the reciprocal value of the highest positivedilution of the test serum, i.e. an anti-VP1 titer or anti-peptide titerof 8 means that antibodies could still be detected at a dilution of thetest serum of 1:2⁸=1:256.

Example 9 Time Course of the Immune Response in Piglents

9.1. Time Courses of the Anti-VP1 Titers

FIG. 7 depicts the time course of the immune responses to the VP1-BC2capsomer and VP1-BC2 capsoid, selected here by way of example, i.e.depicts the time course of the anti-VP1 titers after immunization with:200 μg of VP1-BC2 pentamer (P200); 50 μg of VP1-BC2 pentamer (P50), 200μg of VP1-BC2 capsoid (C200); 50 μg of VP1-BC2 capsoid (C50); 200 μg ofVP1 wild type pentamer (VP1wt, P 200). The average and standarddeviation of each group are indicated (n=4). For each measurement(Pre=before immunization, Wk 2=week 2, Wk 4=week 4, . . . ), thecorresponding immunizations are plotted as bars from left to right, i.e.the first bar from the left is P200, the second bar from the left isP50, etc.

For detection of the anti-VP1 titers, the test was carried out onlyagainst wild type-VP1 pentamers, and therefore peptide-specificantibodies are not included. Anti-VP1-IgG titers of ≦5 were regarded asnegative and were given a score of log 2 titer level 1. While preimmunesera were, without exception, anti-VP1 negative, all animals wereevaluated as anti-VP1 positive 2-6 weeks after labeling. VP1-BC2 inducedhigh immune responses (log 2 titers of 10-12), and even 6 weeks afterimmunization there was still no distinct decrease in the immune responserecordable. Significant differences were found neither between viralcapsomers and capsoids nor between vaccine doses of 200 μg and 50 μg,meaning that it is possible to use viral capsomers for labeling withoutproblems, i.e. the complicated reconstitution/assembly step is dispensedwith.

9.2. Time Courses of the Anti-peptide Titers

FIG. 8 depicts the time course of the immune responses to the peptideinserted into VP1 by way of example, i.e. depicts the time course of theanti-peptide titer after immunization with: 200 μg of VP1-BC2 pentamer(P200); 50 μg of VP1-BC2 pentamer (P50), and 200 μg of VP1-BC2 capsoid(C200); 50 μg of VP1-BC2 capsoid (C50); and 200 μg of VP1 wild typepentamer (VP1wt, P 200). The average and standard deviation of eachgroup are indicated (n=4). For each measurement (Wk 2=week 2, Wk 4=week4, Pre=before immunization), the corresponding immunizations are plottedas bars from left to right, i.e. the first bar on the left is P200, thesecond bar from the left is P50, etc.

For detection of the anti-peptide titers, tests were only carried outagainst the peptide. Anti-peptide-IgG titers of □ 3 were regarded asnegative and given a score of the log 2 titer level of 1. As expected,both the preimmune sera and the animals labeled with VP1 wild type hadanti-peptide-negative immune responses (exception: in each case 1 animalof the VP1 wild type group in weeks 4 and 6). In contrast, all groupsvaccinated with VP1-BC2 were registered as anti-peptide positive 2-6weeks after labeling. The antibodies specifically directed against thepeptide reached log 2 titer levels of up to 9 as a group average (200 μgof capsoid, week 4). In contrast to the capsoids, the capsomers hadanti-peptide titers which were lower by 2-3 titer levels, but they arestill detectable as positive in the ELISA. Reducing the dose here seemsto result in an increase in the immune response.

Overall, the values of the anti-peptide titers are below those of theanti-VP1 titers. Since the total surface of the peptide is distinctlysmaller than that of the carrier capsomer and since the carrier capsomerhas a larger number of different epitopes, such a difference was to beexpected.

9.3 Time Courses of the Anti-VP1 Titers—Comparison: Pentamers with andwithout Boost

FIG. 9 depicts the time course of the immune responses to VP1 capsomersin pigs over a longer period (W0, W3, W7 etc.=week 0, week 3, week 7etc.). The time course of the anti-VP1 titers in pigs after immunizationwith in each case 200 μg of VP1 capsomers with and without boost (inweek 3) is shown. The antigen-specific detection of antibodies wascarried out by means of ELISA on microtiter plates coated with VP1pentamers. In addition, the adjuvant Montanide IMS1313 was used in theimmunization. The average and standard deviation of each group areindicated (n=4). For detection of the anti-VP1 titers, only testsagainst wild type-VP1 were carried out. Anti-VP1-IgG titers of ≦5 wereregarded as negative. It is revealed that the groups without boostimmunization also have a long-lasting immune response. Without boostimmunization, a titer reduction by only 1 titer level (log 2 value) isrecorded within a period of 20 weeks. In both animal groups, anti-VP1antibody titers of 11-13 (log 2 value) are achieved up to week 20. Evenin the meat juice, the label is still clearly detectable in both groups.

The data illustrate that long-term labeling with the composition of theinvention is also possible in pigs.

In summary, this example indicates that labeling based on immunogenicityby administrating viral capsomers is a simple and cost-effective method.The viral capsomers used here elicit a high anti-VP1 immune response. Nosignificant difference with respect to the anti-VP1 titers was foundwhen comparing between the vaccination with capsoids and pentamers. Thusthe costly and complicated step of assembly to capsoids is no longernecessary.

Example 10 Long-term Time Course of the Immune Response in Cattle

10.1 Time Course of the Anti-VP1 Titers—Dose Comparison

FIG. 10 depicts the time course of the immune responses to polyoma VP1capsoids, i.e. the time course of the anti-VP1 titers after immunizationwith: 10 μg or 100 μg of polyoma VP1 capsoid in the presence of theadjuvant Quil A. The average and standard deviation of each group areindicated (n=5). For detection of the anti-VP1 titers, tests were onlycarried out against wild type VP1; therefore, peptide-specificantibodies are not included. Anti-VP1-IgG titers of ≦5 were regarded asnegatives. All animals were evaluated as anti-VP1 positive as early asin week 2 after labeling. The polyoma VP1 capsoid-induced immuneresponses were in the range from 8-10, and remained up to week 20 afterimmunization. Significant differences with regard to the twoconcentrations used were not found. Detection of the antigen-specificantibodies in serum-immunized cattle was carried out by means of ELISAon microtiter plates coated with VP1 pentamers.

10.2 Time Courses of the Anti-VP1 Titers—Comparison: Pentamers—Capsoids

FIG. 11 depicts the time course of the immune responses to VP1 capsoidsand VP1 capsomers in cattle over a relatively long period. The timecourse of the anti-VP1 titers after immunization with in each case 100μg of VP1 capsoid or 200 μg of pentamers (=capsomers) in cattle isdepicted there. The antigen-specific detection of antibodies was carriedout by means of ELISA on microtiter plates coated with pentamers andcapsoids, respectively. In addition, the adjuvant Quil A was used in theimmunization. The average and standard deviation of each group areindicated (n=2). For detection of the anti-VP1 titers, only testsagainst wild type VP1 were carried out. Anti-VP1-IgG titers of ≦5 wereregarded as negative. It is revealed, that the pentamers elicitidentical or higher immune responses than the fully assembled VP2capsoids. These responses remain even in week 24, illustrating that thecomposition of the invention or the method of the invention haveexcellent suitability for long-term labeling.

Example 11 Accelerated Test

FIG. 12 depicts an exemplary embodiment of an accelerated test of theinvention which is suitable for detecting the label(s) in situ. Theantigen (i.e. the viral capsomer or the peptide coupled to bovine serumalbumin [BSA]) is immobilized on an analytical membrane on the test line(T). Downstream of the analytical membrane is a conjugate release regionwhich contains a dried conjugate of gold with antibodies which arespecific for IgG or IgA of the living being to be tested (i.e. cattle,pigs, etc.). Furthermore, the accelerated test has a sample applicationregion. The use is as follows: the liquid sample containing the bodyfluid to be examined in which a possible immune response is to be testedis applied to the sample application region and, owing to capillaryforces, migrates through the conjugate region, whereby the goldconjugate is rehydratized, enabling an interaction between theanti-peptide antibodies or anti-viral capsomer antibodies present in thebody fluid and the gold conjugate, if such antibodies are present in thebody fluid. The complex of gold and the two antibodies migrates to thetest line at which the antigen is located (antigen coupled to BSA oranother carrier), and is immobilized there and generates a colored line.A second line, the “control line”, indicates in each case of a correctlycarried out test a signal which is mediated via a biotin-streptavidinbinding. Biotin is located on the membrane and streptavidin is locatedin the gold conjugate.

The staining of the control line indicates that the accelerated test iscompleted. This test may provide rapid results within 5 minutes.Similarly, “dip sticks” may be constructed which are based on the sameprinciple and in which a sample contact region is contacted with thebody fluid to be studied and in which the subsequent reactions proceedin the same manner as in the accelerated test just described.

The previous examples of labeling with capsomers have shown thatlong-term labeling over 24 weeks is possible without boost, i.e. withoutfurther administration of the antigen. The stability of theanti-capsomer titers is thus extremely high.

By linking immunogenic peptide sequences to the carrier capsomer, amultiplicity of labeling combinations may be used. The direct cloning ofthe peptide sequence(s) into the capsomer as illustrated in Example 2,dispenses with the additional complicated step of conjugating thepeptide/peptides to the capsomer.

The use of a simple accelerated test as described in Example 11, makesusing the composition of the invention or the method of the inventionextremely simple and uncomplicated and thus is a simple way which can beused even by non-experts to carry out the appropriate labeling tests orthe appropriate monitoring.

The features disclosed in the above description, the claims and thedrawings may, both individually and in any combination, be important forimplementing the invention.

1-46. (canceled)
 47. The use of a protein complex for preparing acomposition for labeling living beings, the protein complex being asingle viral capsomer which is not in the form of a viral capsoid and issoluble in aqueous solution, the viral capsomer being producedrecombinantly and being associated with at least one peptide which isimmunogenic when administered to a living being, the at least onepeptide having been inserted recombinantly into the viral capsomer, theviral capsomer being derived from a virus selected from the group ofnon-enveloped viruses, comprising Papovaviridae, Iridoviridae,Adenoviridae, Parvoviridae, Picomaviridae, Caliciviridae, Reoviridae andBimaviridae.
 48. The use as claimed in claim 47, wherein the proteincomplex is a single viral capsomer which is soluble in aqueous solutionand which is an aggregated sandwich with other single viral capsomerssoluble in aqueous solution.
 49. The use as claimed in claim 47, whereinthe Papovaviridae comprise polyoma and papilloma viruses and thePicomaviridae comprise polio viruses.
 50. The use as claimed in claim47, wherein the viral capsomer is derived from polyoma virus, inparticular murine polyoma virus.
 51. The use as claimed in claim 47,wherein the viral capsomer is a pentamer, hexamer or heptamer.
 52. Theuse as claimed in claim 50, wherein the viral capsomer is a pentamer ofmurine polyoma virus VP1 or is a pentamer of murine polyoma virus VP1 inassociation with murine polyoma virus VP2, or is a pentamer of murinepolyoma virus VP1 in association with murine polyoma virus VP3, or is acombination of the aforementioned possibilities.
 53. The use as claimedin claim 52, wherein the viral capsomer is a pentamer of murine polyomavirus VP1.
 54. The use as claimed in claim 47, wherein the viralcapsomer does not derive or cannot be obtained from a virus selectedfrom the group comprising CSF virus (swine fever virus), foot-and-mouthdisease virus, PPV (porcine parvovirus), influenza virus, in particularinfluenza A virus, bovine leukemia virus (EBL virus) (BLV), bovineherpes virus (BHV1), bovine viral diarrhea virus (MD virus), bovinepolyoma virus (BpyV), rotavirus, porcine herpes virus 1, pseudorabiesvirus, PRRS virus and TGE virus.
 55. The use as claimed in claim 47,wherein the association of viral capsomer and peptide is soluble inaqueous solution.
 56. The use as claimed in claim 47, wherein thepeptide is a peptide eliciting a B-cell response.
 57. The use as claimedin claim 47, wherein the peptide has a sequence derived from a virus, aprokaryotic cell or a eukaryotic cell or that the peptide has a sequencewhich is of artificial origin.
 58. The use as claimed in claim 47,wherein the peptide comprises no more than 5-35 amino acids.
 59. The useas claimed in claim 58, wherein the peptide comprises no more than 5-20amino acids.
 60. The use as claimed in claim 59, wherein that thepeptide comprises no more than 5-15 amino acids.
 61. The use as claimedin claim 47, wherein the viral capsomer is derived from a first virusand the peptide is derived from a second virus which is not the same asthe first virus.
 62. The use as claimed in claim 61, wherein the peptideis derived or can be obtained from a virus selected from the group ofnon-enveloped viruses, comprising Papovaviridae, in particular polyomaand papilloma viruses, Iridoviridae, Adenoviridae, Parvoviridae,Picomaviridae, in particular polio viruses, Caliciviridae, Reoviridaeand Bimaviridae.
 63. The use as claimed in claim 62, wherein the peptideis derived or can be obtained from a virus selected from the group ofenveloped viruses, comprising Poxviridae, Herpesviridae, Hepadnaviridae,Retroviridae, Paramyxoviridae, Sendaiviridae, Orthomyxoviridae,Bunyaviridae, Arenaviridae, Toroviridae, Togaviridae, Flaviviridae,Rhabdoviridae and Filoviridae.
 64. The use as claimed in claim 47,wherein the peptide does not derive or cannot be obtained from an agent,for example a virus, bacterium or a eukaryotic cell, which enters theorganism of the living being in the form of a vaccine or medicarnent orvia the food chain or, under normal conditions of life of said livingbeing, via the environment and/or to which antibodies are produced insaid living being under normal conditions of life.
 65. The use asclaimed in claim 64, wherein the peptide does not derive or cannot beobtained from a virus selected from the group comprising CSF virus(swine fever virus), foot-and-mouth disease virus, PPV (porcineparvovirus), influenza virus, in particular influenza A virus, bovineleukemia virus (EBL virus) (BLV), bovine herpes virus (BHV1), bovineviral diarrhea virus (MD virus), bovine polyoma virus (BpyV), rotavirus,porcine herpes virus 1, pseudorabies virus, PRRS virus and TGE virus.66. The use as claimed in claim 65, wherein the peptide does not derivefrom Leptospira, in particular L. grippotyphusa, L. tarassovi, L.canicola, L. pomona, L. bratislava, Chlamydia, in particular C.psittaci, Brucella, in particular B. abortus, B. canis, B. melitensis,Mycobacterium, in particular M. avium subsp. paratuberculosis orCoxiella, in particular C. burnetii.
 67. The use as claimed in claim 47,wherein the peptide is an artificial peptide.
 68. The use as claimed inclaim 47, wherein the at least one peptide has been coexpressed with thecapsomer protein, starting from a DNA encoding said at least one peptideand said capsomer protein.
 69. The use as claimed in claim 47, whereinthe viral capsomer is associated with two or more peptides as defined inany of the preceding claims.
 70. The use as claimed in claim 47, whereinthe viral capsomer and/or the at least one peptide are in the form ofthe nucleic acid coding therefor.
 71. The use as claimed in claim 47,wherein, upon singular administration oi said composition to a livingbeing, the viral capsomer elicits in said living being an immuneresponse which can still be detected at least 18 weeks postadministration.
 72. The use as claimed in claim 71, wherein the immuneresponse can still be detected after at least 20 weeks.
 73. The use asclaimed in claim 72, wherein the immune response can still be detectedat least 24 weeks post administration.
 74. The use as claimed in claim71, wherein the immune response manifests itself in the form of anincreased anti-viral capsomer-IgG and/or -IgA titer and/or an increasedanti-viral capsomer protein-IgG and/or -IgA titer and/or an increasedanti-peptide-IgG and/or -IgA titer.
 75. The use as claimed in claim 74,wherein the increased anti-viral capsomer/viral capsomerprotein/peptide-IgG and/or -IgA titer is at least 1:64.
 76. A method oflabeling living beings or agents administered to living beings,comprising the following steps: a) adding a protein complex as definedin claim 47 to an agent to be labeled, b) administering said agent to aliving being, c) detecting the immunoresponse caused by saidadministration in said living being by means of an enzyme-immunologicalor immunochemical method.
 77. The method as claimed in claim 76, whereinthe immune response comprises a formation of antibodies.
 78. The methodas claimed in claim 77, wherein the antibodies are secreted antibodiesand/or antibodies exposed on lymphocyte surfaces.
 79. The method asclaimed in claim 76, wherein detection takes place in a body fluidselected from the group comprising meat juice, blood, whole blood,plasma, lymph, serum, saliva, milk, urine and semen.
 80. The method asclaimed in claim 78, wherein the lymphocytes are B-lymphocytes and/orB-lymphocytes in combination with T-lymphocytes.
 81. The method asclaimed in claim 76, wherein the administration is carried out once orseveral times, in the latter case at intervals of several weeks.
 82. Themethod as claimed in claim 76, wherein agent is a medicament, a vaccineor stored blood.
 83. The method as claimed in claim 82, wherein theagent is an anti-infectious agent, in particular an antibiotic.
 84. Themethod as claimed in claim 76, wherein the living being is a non-humanmammal.
 85. An antibody directed against the viral capsomer and/or theat least one peptide of the protein complex as defined in claim
 47. 86.An antibody directed against the antibody as claimed in claim
 85. 87. Anaccelerated test comprising the antibody as claimed in claim 86 at leastone of the viral capsomer as defined in claim 47 and/or the peptide asdefined in claim
 47. 88. The accelerated test as claimed in claim 87,wherein the antibody and/or the viral capsomer and/or the peptide arecoupled to a reporter reagent.